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Effects of Opuntia megacantha on blood glucose and kidney function in streptozotocin diabetic rats
Journal of Ethnopharmacology 69 (2000) 247 – 252 www.elsevier.com/locate/jethpharm
Short communication
Effects of Opuntia megacantha on blood glucose and kidney function in streptozotocin diabetic rats P. Bwititi a, C.T. Musabayane b,*, C.F.B. Nhachi c b
a Department of Medical Laboratory Sciences, Faculty of Medicine, Uni6ersity of Zimbabwe, Harare, Zimbabwe Department of Physiology, Faculty of Medicine, Uni6ersity of Zimbabwe, PO Box MP 167, Mount Pleasant, Harare, Zimbabwe c Department of Clinical Pharmacology, Faculty of Medicine, Uni6ersity of Zimbabwe, Harare, Zimbabwe
Received 3 January 1999; received in revised form 30 June 1999; accepted 6 July 1999
Abstract The purpose of the study was to investigate the effects of Opuntia megacantha leaf extracts on blood glucose concentrations and kidney function in normal and streptozotocin (STZ) – diabetic rats. STZ-diabetic and non-diabetic rats were orally administered extracts of O. megacantha leaves (20 mg/100 g body weight) daily for 5 weeks and respective control rats were administered normal saline (0.1 ml/100 mg body weight). Urine volume, urinary outputs of Na+, K+ and creatinine were monitored daily over the 5-week period. Plasma concentrations of Na+, K+, urea and creatinine and the glomerular filtration rate (GFR) as assessed by creatinine clearance were determined after 5 weeks. Plasma glucose concentrations in STZ-diabetic and non-diabetic rats were reduced by the administration of leaf extracts of O. megacantha. However, leaf extracts increased urinary Na+ output in STZ-diabetic and non-diabetic rats, concomitantly with a reduction in plasma concentration of the ion. O. megacantha leaf extracts significantly increased plasma creatinine and urea concentrations in non-diabetic and STZ-diabetic rats. Administration of the leaf extract was also associated with an increased GFR in STZ-diabetic rats (from 1.8 90.3 ml/min to 2.8 9 0.3 ml/min, n =8) although the rate was unaltered in non-diabetic rats. The results suggest that leaf extracts of O. megacantha not only reduce blood glucose levels, but may be toxic to the kidney as shown by the elevation in plasma urea and creatinine concentrations and the reduction of plasma Na+ concentration. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Diabetes mellitus; Streptozotocin; Renal function; Opuntia megacantha
1. Introduction * Corresponding author. Tel.: +263-4-333678; fax: +2634-333678 or 333407. E-mail address: [email protected] (C.T. Musabayane)
Extracts from Aloe barbadensis, Solanum 6erbascifolum and Opuntia streptacantha are used in
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 1 2 3 - 3
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the treatment of diabetes mellitus in developing countries (Lin, 1972; Frati-Munari et al., 1989, 1990; Ajabnoor, 1990; Roman-Ramos et al., 1991; Frati et al., 1991). The mechanisms through which these plant extracts exert hypoglycaemic effects are not clear. O. streptacantha of the same genus of O. megacantha has been previously established to exert hypoglycaemic effects (Frati-Munari et al., 1989). Therefore, we investigated the effects of O. megacantha on plasma insulin and glucose concentrations in normal rats and streptozotocin (STZ)-induced diabetic rats. Since streptozotocin selectively destroys b-cells of the pancreas to induce insulin-dependent diabetes (Grussner et al., 1993), we postulated that extracts from the plant would not affect blood glucose levels in STZ-diabetic rats if the mode of action is mediated through insulin production. We also studied the effects of O. megacantha leaf extracts on renal function to establish whether the plant extracts can reverse the previously reported inability of the kidney to excrete Na+ in STZ-diabetis mellitus (Musabayane et al., 1995). Kidney function can be divided into: effective plasma flow, glomerular filtration rate (GFR) and the ability to concentrate or dilute urine and tubular function. However, GFR is a fundamental parameter of kidney function, evaluating renal function and creatinine clearance test is used in rats to assess GFR (Bursztyn et al., 1995; Travlos et al., 1996). Therefore, the current study also monitored creatinine clearance rates in relationship to urinary excretion outputs and plasma urea concentrations in control and treated rats to establish the effects of O. megacantha leaf extracts on kidney function.
2. Methods and materials
2.1. Preparation of O. megacantha leaf extract O. megacantha plants were collected around Harare, Zimbabwe and authenticated at the National Herbarium, where voucher specimens were deposited (number 5417). The leaves were homogenized using a blender and dried for 48 h at 40°C. Plant homogenate (100 g) was mixed with 150 ml of 80% ethanol and stirred for 4 h and filtered.
The filtrate was dried in an evaporator and a yield of 1.57% of the prepared plant material was dissolved in normal saline.
2.2. Animals Male Sprague–Dawley rats (250–300 g) bred and housed in the Medical Faculty Animal House, University of Zimbabwe were used. Rats were made diabetic by an intraperitoneal (i.p.) injection of streptozotocin (STZ 60 mg/kg) in citrate buffer, pH 6.3. Animals that exhibited glucosuria after 24 h, tested by the Combur 9 test (Boehringer, Germany) were considered diabetic. Animals injected with citrate buffer acted as nondiabetic controls. The rats were kept in separate metabolism cages (NPK, cages, Dartford, Kent, UK) that were cleaned daily. The diabetic rats were divided into two groups, one orally administered saline daily (0.1 ml/100 mg body weight, n= 8) and the other orally administered daily, leaf extracts of O. megacantha leaves (20 mg/100 g body weight, n= 8). Non-diabetic rats injected with citrate buffer were similarly divided into treated and untreated groups. The extract and saline were administered daily at 09:00 h for 5 weeks by means of a bulbeb steel tube. The rats were given both food (Mouse Comproids, National Foods, Harare) and water ad libitum. Amounts consumed and body weights were recorded daily for 5 weeks. Urine volume and urinary concentrations of Na+ and K+ were determined daily at 09:00 h for 5 weeks.
2.3. Measurements Kidney weights were determined in all groups of rats after 5 weeks. Creatinine clearance determined after 5 weeks involved 24-h urine collection and 0.1 ml blood collected from the tail vein simultaneously to allow calculation of creatinine clearance as a measure of GFR. The fractional excretion rates of sodium (FENa+) and potassium (FEK+) were calculated. After 5 weeks, blood was collected by decapitation of animals from all groups into pre-cooled fluoride tubes for glucose and in heparin tubes for plasma creatinine, Na+ and K+ determinations.
P. Bwititi et al. / Journal of Ethnopharmacology 69 (2000) 247–252
Blood for insulin was collected into plain tubes and the separated serum was stored in a Bio Ultra freezer (Mallkinckrodt, OH) at −70°C until assayed. The measurements were done by radioimmunoassay, Amerlex TM-M magnetic separation (Amersham International plc, Amersham, UK) and counts read on a LKB 1261 multigamma counter (Wallac, Finland). Plasma glucose concentrations were measured by the glucose oxidase method immediately after collection. Creatinine estimation employed the reaction of creatinine and sodium picrate to form creatinine picrate. Urea estimation employed the hydrolytic degradation of urea in the presence of urease. The methods used reagent kits from Biotrol (Biotrol Pathologique, France) and assays were done on a Cobas Mira S (Roche, France). Sodium and potassium concentrations were determined by flame photometry (Instrument Laboratory, USA).
2.4. Analysis of data Values are presented as means 9S.E.M. Kidney function was evaluated by the creatinine clearance test as assessed by 24-h urinary excretion rates of creatinine in relation to plasma concentration. Renal function was also assessed by calculating total weekly; fluid voided and urinary amounts of electrolytes excreted. To determine the effects of the leaf extract of O. megacantha, the data were treated and presented separately for the non-diabetic and STZ-diabetic rats. The data
249
were subjected to analysis of variance using a one-way design and Scheffes multiple comparison was used to assess any differences. A value of PB 0.05 was considered significant.
3. Results
3.1. Water and electrolyte turno6er Table 1 compares body weight changes, food and water intake and total fluid voided non-diabetic and STZ-diabetic rats administered O. megacantha leaf extracts daily over the 5-week period with respective control groups. Non-diabetic rats gained weight throughout the 5-week period, but the administration of O. megacantha leaf extract significantly (PB0.01) reduced the magnitude of weight gain. However, the leaf extracts did not affect body weight in STZ-diabetic rats which significantly lost weight over the 5week study period. STZ-diabetic animals drank more water by comparison with non-diabetic animals. However, administration of the extracts increased water intake in non-diabetic and STZ-diabetic rats. Administration of O. megacantha leaf extracts significantly (PB0.01) reduced food intake in non-diabetic and diabetic rats. Kidney weight was significantly increased in STZ-diabetic rats by comparison with non-diabetic animals. Administration of the O. megacantha leaf extracts did not affect the kidney mass
Table 1 Comparison of percentage weight change, kidney mass, 5-week measures of food and water taken and fluid voided in control non-diabetic and STZ-diabetic rats and non-diabetic and STZ-diabetic rats administered O. megacantha leaf extract (n = 8 in all groups) Anaysis
Weight change (%) Food taken (g) Water taken (ml) Fluid voided (ml) Kidney mass (g/100 g body weight)
Non-diabetic
Diabetic
Untreated
Treated
Untreated
Treated
+139 1 5419 28 8319 38 4429 34 0.37 9 0.06
+991* 457 925* 991 937* 469 932 0.45 90.07
−169 1** 927 9 31** 3661 9 129** 3084 9 135** 0.92 90.04**
−1692 741 960* 4008 9103* 3274 997 0.89 90.07
* PB0.05 by comparison with respective untreated rats. ** PB0.05 by comparison with untreated non-diabetic rats.
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Table 2 Kidney mass, plasma creatinine, glucose, Na+ and K+ concentrations, urinary Na+, K+ and creatinine excretion, FENa+ and FEK+ rates over 5 weeks in control, non-diabetic and STZ-diabetic rats and non-diabetic and STZ-diabetic rats administered O. megacantha leaf extract (n =8 in all groups) Analysis
Plasma [Na+] (mmol/l) FeNa+ (%) UrinaryNa+ excreted (mmol/l/24 h) Plasma [K+] (mmol/l) FEK+ (%) UK+ excreted (mmol/l/24 h) Plasma [urea] (mmol/l) Plasma [creatinine] (mmol/l) Urinary creatinine excreted/ml/24 h Glucose (mmol/l) Insulin (ng/ml)
Non-diabetic
Diabetic
Untreated
Treated
Untreated
Treated
137 91 0.459 0.04 7519 41 4.929 0.30 1.59 0.5 6549 17 6.39 3 29 9 2 4.29 0.2 7.7 9 0.2 13.69 0.6
134 9 1* 1.15 9 0.02* 1203 9 46* 4.53 90.20 1.1 90.3 634 985 7.4 90.4* 37 92* 4.8 90.2 6.1 90.1** 12.6 90.7
142 9 1 1.40 90.04 359 938** 3.61 90.51** 9.4 91.3** 1054 9 82** 11.2 9 0.1** 47 9 2** 1.8 9 0.3** 35.4 9 0.6** 1.3 9 0.1**
133 9 1* 1.40 90.03 531 935* 3.72 90.32 11.49 0.5* 954 9 67* 16.0 9 0.9* 55 9 3* 2.8 9 0.3* 30.7 9 0.7* 1.5 9 0.2
* PB0.05 by comparison with respective untreated rats. ** PB0.05 by comparison with non-diabetic rats.
in non-diabetic and STZ-diabetic rats (Table 1). Preliminary studies showed that O. megacantha leaf extracts were more potent in lowering blood glucose concentrations.
3.2. Renal function Glycosuria, polyuria and diarrhoea were observed from the first week in STZ-diabetic rats and were associated with decreased total urinary output of Na+ over the 5-week period (Table 2). Administration of O. megacantha leaf extracts significantly reduced plasma Na+ concentrations in non-diabetic and STZ-diabetic rats. FENa+ was significantly (PB0.05) increased in non-diabetic animals administered O. megacantha leaf extracts. However, plasma concentrations of Na+ did not statistically differ between non-diabetic and STZdiabetic rats (Table 2). O. megacantha leaf extracts administration significantly (P B0.01) increased plasma urea and creatinine concentrations in non-diabetic and STZ-diabetic rats (Table 2). A significant increase in creatinine clearance rates were observed in STZ-diabetic rats, but the rates were not altered in non-diabetic rats (Table 2).
The total amounts of urinary K+ in STZ-diabetic rats was significantly higher than in non-diabetic rats (Table 2). This resulted in a significantly low plasma concentration in STZ-diabetic animals by comparison with non-diabetic animals. Administration of O. megacantha leaf extracts caused significant reduction in urinary K+ output in STZ-diabetic rats. The urinary K+ output in nondiabetic was slightly lowered, but did not reach statistically significance. Administration of leaf extracts also increased the FEK+ rate in STZ-diabetic rats, but did not have any effect in non-diabetic animals.
3.3. Plasma insulin and glucose le6els Plasma glucose levels were significantly (PB 0.05) elevated in STZ-diabetic rats and this was associated with low plasma insulin concentrations by comparison with non-diabetic rats (Table 2). Administration of O. megacantha leaf extracts over the 5-week period significantly reduced plasma glucose concentration in non-diabetic and STZ-diabetic rats. The plasma insulin concentration was not statistically altered by O. megacantha treatment (Table 2).
P. Bwititi et al. / Journal of Ethnopharmacology 69 (2000) 247–252
4. Discussion Oral administration of O. megacantha leaf extracts daily to non-diabetic and diabetic rats was associated with a reduction in plasma glucose concentrations (Table 2) without affecting basal insulin levels. Preliminary studies showed that oral administration of O. megacantha leaf extracts at various dose rates (10 – 30 mg/100 g body weight) significantly reduced blood glucose concentrations in a pattern that could not be separated. Therefore, the median dose (20 mg/100 g body weight) was chosen for the current study. Since streptozotocin selectively destroys b-cells of the pancreas, we would expect the extracts to exert no effect on plasma glucose concentrations in STZ-diabetic rats if the mode of action is mediated through insulin production. However, our results show that the extract decreased plasma glucose without affecting the basal insulin levels in diabetic and non-diabetic rats. Therefore, these results suggest that the hypoglycaemic effects observed appear to involve mechanisms that do not involve insulin. We suggest that the extract promoted glucose entry into cells accompanied by Na+ reabsorption. Using boiled stem of O. streptacantha Frati et al. (1991) reported significant decrease in serum glucose levels in normal humans and type 2 diabetic subjects. In both cases C-peptide, which is an index of insulin production, remained unchanged (Frati et al., 1991). This agrees with the present study since leaf extract did not change plasma insulin levels. The results of the current study suggest that oral administration of leaf extract of O. megacantha at 20 mg/100 g body weight produces toxic effects on the kidneys as shown by the elevation in plasma creatinine and urea concentration in diabetic and non-diabetic rats (Table 2). Treatment-related increases in plasma creatinine and urea concentrations are variables used not only to indicate impairment of kidney function (Braunlich et al., 1997; Hwang et al., 1997), but are also clinical chemistry end points to detect treatmentrelated toxic effects of compounds on the kidney in rats (Travlos et al., 1996). Indeed, a relationship between treatment-related alterations in plasma urea and creatinine concentrations and
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histopathology of the kidney has been reported in rats (Travlos et al., 1996). Thus, the observed changes in renal function may be mediated via O. megacantha leaf extracts induced alterations in kidney structure. O. megacantha leaf extracts increased 24 h urinary creatinine clearance in STZ-diabetic rats, but there was no change in non-diabetic rats. If the extract produced toxic effects on the kidney to increase urinary creatinine clearance in STZ-diabetic rats, then we should also expect a rise in urinary creatinine excretion in non-diabetic rats administered O. megacantha leaf extract. The differences can possibly be attributed to differences in kidney mass between diabetic and non-diabetic rats. STZ-diabetic rats had increased kidney weight by comparison with non-diabetic animals. Typically, increased kidney size accompanies elevation in GFR because of increased glomerular volume and capillary surface area (Yamada et al., 1992). Therefore, we cannot exclude the involvement of changes in filtration surface area and elevated plasma creatinine concentration on the observed changes in creatinine clearance in STZdiabetic rats. The urinary creatinine excretion of non-diabetic rats administered the extract over 5 weeks showed a mean rise of 10% and 20% over that measured in untreated non-diabetic animals (Table 2). We suggest that this elevation in urinary clearance of creatinine following administration of O. megacantha leaf extracts is of biological significance indicating early stages of kidney dysfunction. This possibly contributed to the natriuresis observed in treated non-diabetic rats. O. megacantha significantly (P B 0.05) increased the FENa+ in non-diabetic animals, but the rate was not altered in STZ-diabetic rats possibly because of enhanced glucose/sodium co-transport in the proximal tubule in these animals. Indeed, administration of O. megacantha leaf extracts was associated with a reduction in plasma Na+ concentrations in non-diabetic and STZ-diabetic rats. In summary, the results of this study suggest that leaf extracts of O. megacantha may be used to manage mild diabetes mellitus. However, further studies are needed to establish how O. megacantha induces kidney toxicity leading to the
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impairment of kidney function as shown by an elevation in plasma urea and creatinine concentrations.
Acknowledgements This study was supported by research grant number YYHO1O/3781 from the Research Board, University of Zimbabwe.
References Ajabnoor, M.A., 1990. Effect of aloes on blood glucose levels in normal and alloxan diabetic mice. J. Ethnopharmacol. 28 (2), 215 – 220. Braunlich, H., Marx, F., Fleck, C., Stein, G., 1997. Kidney function in rats after 5/6 nephrectomy (5/6 NX); effort of treatment with vitamin E. Exp. Toxicol. Pathol. 49 (1–2), 135 – 139. Bursztyn, M., Ben-Ishay, D., Mekler, J., Raz, I., 1995. Insulininduced renal dysfunction in regular Sabra rats. Clin. Exp. Pharmacol. 22 (1), S32–S33. Frati-Munari, A.C., de Leon, C., Ariza-Andraca, R., BanalesHam, M.B., Lopez-Ledesma, R., Lozoya, X., 1989. Effect of a dehydrated extract of nopal (Opuntia ficus indica Mill.) on blood glucose. Arch. Invest. Med. 20, 211–216.
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Frati-Munari, A.C., Licona-Quesada, R., Ariza-Andraca, C.R., Lopez-Ledesma, R., Chavez-Negrete, A., 1990. Activity of Opuntia streptacantha in healthy individuals with induced hyperglycaemia. Arch. Invest. Med. 21, 99 – 102. Frati, A.C., Xilotl Diaz, N., Altmirano, P., Ariza, R., LopezLedesma, R., 1991. The effects of two sequential doses of Opuntia streptacantha upon glycaemia. Arch. Invest. Med. 22, 333 – 336. Grussner, R., Nakhleh, R., Grussner, A., Tomadze, G., Diem, P., Sutherland, D., 1993. Streptozotocin-induced diabetes in pigs. Horm. Metab. Res. 25 (4), 199 – 203. Hwang, D.F., Lai, Y.S., Chiang, M.T., 1997. Toxic effects of grass carp, snake and chicken bile juices in rats. Toxicol. Lett. 85 (2), 85 – 92. Lin, C.C., 1972. Crude drugs used for the treatment of diabetes in Taiwan. Am. J. Clin. Med. 20 (304), 269 – 279. Musabayane, C.T., Ndhlovu, C.E., Balment, R.J., 1995. Renal fluid and electrolyte handling in streptozotocin-diabetic rats. Renal Failure 17, 107 – 116. Roman-Ramos, R., Flores-Saenz, J.L., Partida-Harnandez, G., Lara-Lemus, A., Alarcon-Aguilar, F., 1991. Experimental study of the hypoglycaemic effects of some antidiabetic plants. Arch. Invest. Med. 22, 87 – 93. Travlos, G.S., Morris, R.W., Elwell, M.R., Duke, A., Rosenblum, S., Thompson, M.B., 1996. Frequency and relationship of clinical chemistry and liver and kidney histopathology findings in 13-week toxicity studies in rats. Toxicology 107 (1), 17 – 29. Yamada, H., Hishida, A., Kumagai, H., Nishi, S., 1992. Effects of age, renal diseases and diabetes mellitus on renal size reduction accompanied by the decrease on renal function. Nippon Jinzo Gakkai Shi 34 (10), 1071 – 1075.
Short communication
Effects of Opuntia megacantha on blood glucose and kidney function in streptozotocin diabetic rats P. Bwititi a, C.T. Musabayane b,*, C.F.B. Nhachi c b
a Department of Medical Laboratory Sciences, Faculty of Medicine, Uni6ersity of Zimbabwe, Harare, Zimbabwe Department of Physiology, Faculty of Medicine, Uni6ersity of Zimbabwe, PO Box MP 167, Mount Pleasant, Harare, Zimbabwe c Department of Clinical Pharmacology, Faculty of Medicine, Uni6ersity of Zimbabwe, Harare, Zimbabwe
Received 3 January 1999; received in revised form 30 June 1999; accepted 6 July 1999
Abstract The purpose of the study was to investigate the effects of Opuntia megacantha leaf extracts on blood glucose concentrations and kidney function in normal and streptozotocin (STZ) – diabetic rats. STZ-diabetic and non-diabetic rats were orally administered extracts of O. megacantha leaves (20 mg/100 g body weight) daily for 5 weeks and respective control rats were administered normal saline (0.1 ml/100 mg body weight). Urine volume, urinary outputs of Na+, K+ and creatinine were monitored daily over the 5-week period. Plasma concentrations of Na+, K+, urea and creatinine and the glomerular filtration rate (GFR) as assessed by creatinine clearance were determined after 5 weeks. Plasma glucose concentrations in STZ-diabetic and non-diabetic rats were reduced by the administration of leaf extracts of O. megacantha. However, leaf extracts increased urinary Na+ output in STZ-diabetic and non-diabetic rats, concomitantly with a reduction in plasma concentration of the ion. O. megacantha leaf extracts significantly increased plasma creatinine and urea concentrations in non-diabetic and STZ-diabetic rats. Administration of the leaf extract was also associated with an increased GFR in STZ-diabetic rats (from 1.8 90.3 ml/min to 2.8 9 0.3 ml/min, n =8) although the rate was unaltered in non-diabetic rats. The results suggest that leaf extracts of O. megacantha not only reduce blood glucose levels, but may be toxic to the kidney as shown by the elevation in plasma urea and creatinine concentrations and the reduction of plasma Na+ concentration. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Diabetes mellitus; Streptozotocin; Renal function; Opuntia megacantha
1. Introduction * Corresponding author. Tel.: +263-4-333678; fax: +2634-333678 or 333407. E-mail address: [email protected] (C.T. Musabayane)
Extracts from Aloe barbadensis, Solanum 6erbascifolum and Opuntia streptacantha are used in
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 1 2 3 - 3
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P. Bwititi et al. / Journal of Ethnopharmacology 69 (2000) 247–252
the treatment of diabetes mellitus in developing countries (Lin, 1972; Frati-Munari et al., 1989, 1990; Ajabnoor, 1990; Roman-Ramos et al., 1991; Frati et al., 1991). The mechanisms through which these plant extracts exert hypoglycaemic effects are not clear. O. streptacantha of the same genus of O. megacantha has been previously established to exert hypoglycaemic effects (Frati-Munari et al., 1989). Therefore, we investigated the effects of O. megacantha on plasma insulin and glucose concentrations in normal rats and streptozotocin (STZ)-induced diabetic rats. Since streptozotocin selectively destroys b-cells of the pancreas to induce insulin-dependent diabetes (Grussner et al., 1993), we postulated that extracts from the plant would not affect blood glucose levels in STZ-diabetic rats if the mode of action is mediated through insulin production. We also studied the effects of O. megacantha leaf extracts on renal function to establish whether the plant extracts can reverse the previously reported inability of the kidney to excrete Na+ in STZ-diabetis mellitus (Musabayane et al., 1995). Kidney function can be divided into: effective plasma flow, glomerular filtration rate (GFR) and the ability to concentrate or dilute urine and tubular function. However, GFR is a fundamental parameter of kidney function, evaluating renal function and creatinine clearance test is used in rats to assess GFR (Bursztyn et al., 1995; Travlos et al., 1996). Therefore, the current study also monitored creatinine clearance rates in relationship to urinary excretion outputs and plasma urea concentrations in control and treated rats to establish the effects of O. megacantha leaf extracts on kidney function.
2. Methods and materials
2.1. Preparation of O. megacantha leaf extract O. megacantha plants were collected around Harare, Zimbabwe and authenticated at the National Herbarium, where voucher specimens were deposited (number 5417). The leaves were homogenized using a blender and dried for 48 h at 40°C. Plant homogenate (100 g) was mixed with 150 ml of 80% ethanol and stirred for 4 h and filtered.
The filtrate was dried in an evaporator and a yield of 1.57% of the prepared plant material was dissolved in normal saline.
2.2. Animals Male Sprague–Dawley rats (250–300 g) bred and housed in the Medical Faculty Animal House, University of Zimbabwe were used. Rats were made diabetic by an intraperitoneal (i.p.) injection of streptozotocin (STZ 60 mg/kg) in citrate buffer, pH 6.3. Animals that exhibited glucosuria after 24 h, tested by the Combur 9 test (Boehringer, Germany) were considered diabetic. Animals injected with citrate buffer acted as nondiabetic controls. The rats were kept in separate metabolism cages (NPK, cages, Dartford, Kent, UK) that were cleaned daily. The diabetic rats were divided into two groups, one orally administered saline daily (0.1 ml/100 mg body weight, n= 8) and the other orally administered daily, leaf extracts of O. megacantha leaves (20 mg/100 g body weight, n= 8). Non-diabetic rats injected with citrate buffer were similarly divided into treated and untreated groups. The extract and saline were administered daily at 09:00 h for 5 weeks by means of a bulbeb steel tube. The rats were given both food (Mouse Comproids, National Foods, Harare) and water ad libitum. Amounts consumed and body weights were recorded daily for 5 weeks. Urine volume and urinary concentrations of Na+ and K+ were determined daily at 09:00 h for 5 weeks.
2.3. Measurements Kidney weights were determined in all groups of rats after 5 weeks. Creatinine clearance determined after 5 weeks involved 24-h urine collection and 0.1 ml blood collected from the tail vein simultaneously to allow calculation of creatinine clearance as a measure of GFR. The fractional excretion rates of sodium (FENa+) and potassium (FEK+) were calculated. After 5 weeks, blood was collected by decapitation of animals from all groups into pre-cooled fluoride tubes for glucose and in heparin tubes for plasma creatinine, Na+ and K+ determinations.
P. Bwititi et al. / Journal of Ethnopharmacology 69 (2000) 247–252
Blood for insulin was collected into plain tubes and the separated serum was stored in a Bio Ultra freezer (Mallkinckrodt, OH) at −70°C until assayed. The measurements were done by radioimmunoassay, Amerlex TM-M magnetic separation (Amersham International plc, Amersham, UK) and counts read on a LKB 1261 multigamma counter (Wallac, Finland). Plasma glucose concentrations were measured by the glucose oxidase method immediately after collection. Creatinine estimation employed the reaction of creatinine and sodium picrate to form creatinine picrate. Urea estimation employed the hydrolytic degradation of urea in the presence of urease. The methods used reagent kits from Biotrol (Biotrol Pathologique, France) and assays were done on a Cobas Mira S (Roche, France). Sodium and potassium concentrations were determined by flame photometry (Instrument Laboratory, USA).
2.4. Analysis of data Values are presented as means 9S.E.M. Kidney function was evaluated by the creatinine clearance test as assessed by 24-h urinary excretion rates of creatinine in relation to plasma concentration. Renal function was also assessed by calculating total weekly; fluid voided and urinary amounts of electrolytes excreted. To determine the effects of the leaf extract of O. megacantha, the data were treated and presented separately for the non-diabetic and STZ-diabetic rats. The data
249
were subjected to analysis of variance using a one-way design and Scheffes multiple comparison was used to assess any differences. A value of PB 0.05 was considered significant.
3. Results
3.1. Water and electrolyte turno6er Table 1 compares body weight changes, food and water intake and total fluid voided non-diabetic and STZ-diabetic rats administered O. megacantha leaf extracts daily over the 5-week period with respective control groups. Non-diabetic rats gained weight throughout the 5-week period, but the administration of O. megacantha leaf extract significantly (PB0.01) reduced the magnitude of weight gain. However, the leaf extracts did not affect body weight in STZ-diabetic rats which significantly lost weight over the 5week study period. STZ-diabetic animals drank more water by comparison with non-diabetic animals. However, administration of the extracts increased water intake in non-diabetic and STZ-diabetic rats. Administration of O. megacantha leaf extracts significantly (PB0.01) reduced food intake in non-diabetic and diabetic rats. Kidney weight was significantly increased in STZ-diabetic rats by comparison with non-diabetic animals. Administration of the O. megacantha leaf extracts did not affect the kidney mass
Table 1 Comparison of percentage weight change, kidney mass, 5-week measures of food and water taken and fluid voided in control non-diabetic and STZ-diabetic rats and non-diabetic and STZ-diabetic rats administered O. megacantha leaf extract (n = 8 in all groups) Anaysis
Weight change (%) Food taken (g) Water taken (ml) Fluid voided (ml) Kidney mass (g/100 g body weight)
Non-diabetic
Diabetic
Untreated
Treated
Untreated
Treated
+139 1 5419 28 8319 38 4429 34 0.37 9 0.06
+991* 457 925* 991 937* 469 932 0.45 90.07
−169 1** 927 9 31** 3661 9 129** 3084 9 135** 0.92 90.04**
−1692 741 960* 4008 9103* 3274 997 0.89 90.07
* PB0.05 by comparison with respective untreated rats. ** PB0.05 by comparison with untreated non-diabetic rats.
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Table 2 Kidney mass, plasma creatinine, glucose, Na+ and K+ concentrations, urinary Na+, K+ and creatinine excretion, FENa+ and FEK+ rates over 5 weeks in control, non-diabetic and STZ-diabetic rats and non-diabetic and STZ-diabetic rats administered O. megacantha leaf extract (n =8 in all groups) Analysis
Plasma [Na+] (mmol/l) FeNa+ (%) UrinaryNa+ excreted (mmol/l/24 h) Plasma [K+] (mmol/l) FEK+ (%) UK+ excreted (mmol/l/24 h) Plasma [urea] (mmol/l) Plasma [creatinine] (mmol/l) Urinary creatinine excreted/ml/24 h Glucose (mmol/l) Insulin (ng/ml)
Non-diabetic
Diabetic
Untreated
Treated
Untreated
Treated
137 91 0.459 0.04 7519 41 4.929 0.30 1.59 0.5 6549 17 6.39 3 29 9 2 4.29 0.2 7.7 9 0.2 13.69 0.6
134 9 1* 1.15 9 0.02* 1203 9 46* 4.53 90.20 1.1 90.3 634 985 7.4 90.4* 37 92* 4.8 90.2 6.1 90.1** 12.6 90.7
142 9 1 1.40 90.04 359 938** 3.61 90.51** 9.4 91.3** 1054 9 82** 11.2 9 0.1** 47 9 2** 1.8 9 0.3** 35.4 9 0.6** 1.3 9 0.1**
133 9 1* 1.40 90.03 531 935* 3.72 90.32 11.49 0.5* 954 9 67* 16.0 9 0.9* 55 9 3* 2.8 9 0.3* 30.7 9 0.7* 1.5 9 0.2
* PB0.05 by comparison with respective untreated rats. ** PB0.05 by comparison with non-diabetic rats.
in non-diabetic and STZ-diabetic rats (Table 1). Preliminary studies showed that O. megacantha leaf extracts were more potent in lowering blood glucose concentrations.
3.2. Renal function Glycosuria, polyuria and diarrhoea were observed from the first week in STZ-diabetic rats and were associated with decreased total urinary output of Na+ over the 5-week period (Table 2). Administration of O. megacantha leaf extracts significantly reduced plasma Na+ concentrations in non-diabetic and STZ-diabetic rats. FENa+ was significantly (PB0.05) increased in non-diabetic animals administered O. megacantha leaf extracts. However, plasma concentrations of Na+ did not statistically differ between non-diabetic and STZdiabetic rats (Table 2). O. megacantha leaf extracts administration significantly (P B0.01) increased plasma urea and creatinine concentrations in non-diabetic and STZ-diabetic rats (Table 2). A significant increase in creatinine clearance rates were observed in STZ-diabetic rats, but the rates were not altered in non-diabetic rats (Table 2).
The total amounts of urinary K+ in STZ-diabetic rats was significantly higher than in non-diabetic rats (Table 2). This resulted in a significantly low plasma concentration in STZ-diabetic animals by comparison with non-diabetic animals. Administration of O. megacantha leaf extracts caused significant reduction in urinary K+ output in STZ-diabetic rats. The urinary K+ output in nondiabetic was slightly lowered, but did not reach statistically significance. Administration of leaf extracts also increased the FEK+ rate in STZ-diabetic rats, but did not have any effect in non-diabetic animals.
3.3. Plasma insulin and glucose le6els Plasma glucose levels were significantly (PB 0.05) elevated in STZ-diabetic rats and this was associated with low plasma insulin concentrations by comparison with non-diabetic rats (Table 2). Administration of O. megacantha leaf extracts over the 5-week period significantly reduced plasma glucose concentration in non-diabetic and STZ-diabetic rats. The plasma insulin concentration was not statistically altered by O. megacantha treatment (Table 2).
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4. Discussion Oral administration of O. megacantha leaf extracts daily to non-diabetic and diabetic rats was associated with a reduction in plasma glucose concentrations (Table 2) without affecting basal insulin levels. Preliminary studies showed that oral administration of O. megacantha leaf extracts at various dose rates (10 – 30 mg/100 g body weight) significantly reduced blood glucose concentrations in a pattern that could not be separated. Therefore, the median dose (20 mg/100 g body weight) was chosen for the current study. Since streptozotocin selectively destroys b-cells of the pancreas, we would expect the extracts to exert no effect on plasma glucose concentrations in STZ-diabetic rats if the mode of action is mediated through insulin production. However, our results show that the extract decreased plasma glucose without affecting the basal insulin levels in diabetic and non-diabetic rats. Therefore, these results suggest that the hypoglycaemic effects observed appear to involve mechanisms that do not involve insulin. We suggest that the extract promoted glucose entry into cells accompanied by Na+ reabsorption. Using boiled stem of O. streptacantha Frati et al. (1991) reported significant decrease in serum glucose levels in normal humans and type 2 diabetic subjects. In both cases C-peptide, which is an index of insulin production, remained unchanged (Frati et al., 1991). This agrees with the present study since leaf extract did not change plasma insulin levels. The results of the current study suggest that oral administration of leaf extract of O. megacantha at 20 mg/100 g body weight produces toxic effects on the kidneys as shown by the elevation in plasma creatinine and urea concentration in diabetic and non-diabetic rats (Table 2). Treatment-related increases in plasma creatinine and urea concentrations are variables used not only to indicate impairment of kidney function (Braunlich et al., 1997; Hwang et al., 1997), but are also clinical chemistry end points to detect treatmentrelated toxic effects of compounds on the kidney in rats (Travlos et al., 1996). Indeed, a relationship between treatment-related alterations in plasma urea and creatinine concentrations and
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histopathology of the kidney has been reported in rats (Travlos et al., 1996). Thus, the observed changes in renal function may be mediated via O. megacantha leaf extracts induced alterations in kidney structure. O. megacantha leaf extracts increased 24 h urinary creatinine clearance in STZ-diabetic rats, but there was no change in non-diabetic rats. If the extract produced toxic effects on the kidney to increase urinary creatinine clearance in STZ-diabetic rats, then we should also expect a rise in urinary creatinine excretion in non-diabetic rats administered O. megacantha leaf extract. The differences can possibly be attributed to differences in kidney mass between diabetic and non-diabetic rats. STZ-diabetic rats had increased kidney weight by comparison with non-diabetic animals. Typically, increased kidney size accompanies elevation in GFR because of increased glomerular volume and capillary surface area (Yamada et al., 1992). Therefore, we cannot exclude the involvement of changes in filtration surface area and elevated plasma creatinine concentration on the observed changes in creatinine clearance in STZdiabetic rats. The urinary creatinine excretion of non-diabetic rats administered the extract over 5 weeks showed a mean rise of 10% and 20% over that measured in untreated non-diabetic animals (Table 2). We suggest that this elevation in urinary clearance of creatinine following administration of O. megacantha leaf extracts is of biological significance indicating early stages of kidney dysfunction. This possibly contributed to the natriuresis observed in treated non-diabetic rats. O. megacantha significantly (P B 0.05) increased the FENa+ in non-diabetic animals, but the rate was not altered in STZ-diabetic rats possibly because of enhanced glucose/sodium co-transport in the proximal tubule in these animals. Indeed, administration of O. megacantha leaf extracts was associated with a reduction in plasma Na+ concentrations in non-diabetic and STZ-diabetic rats. In summary, the results of this study suggest that leaf extracts of O. megacantha may be used to manage mild diabetes mellitus. However, further studies are needed to establish how O. megacantha induces kidney toxicity leading to the
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impairment of kidney function as shown by an elevation in plasma urea and creatinine concentrations.
Acknowledgements This study was supported by research grant number YYHO1O/3781 from the Research Board, University of Zimbabwe.
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